Andreas Mas Marques

Rotavirus vaccine effectiveness and case-control study on risk factors for breakthrough infections in Germany, 2010-2011.

Background: In the German federal state Mecklenburg-Western Pomerania, routine rotavirus (RV) vaccination in infants has been recommended since 2009. The effectiveness of RV vaccination was investigated after an unexpectedly high number of RV infections in fully vaccinated children occurred. Methods: Intensified RV surveillance was performed in Mecklenburg-Western Pomerania between 2010 and 2011. The screening method was applied to assess vaccine effectiveness (VE) in children up to 24 months after vaccination. To identify risk factors for breakthrough infections, a case-control study and genotyping were conducted in vaccinated and unvaccinated RV-infected children. Results: VE for the prevention of RV infection requiring medical attention or hospitalization was 68% (95% confidence interval [CI]: 61–71) and 80% (95% CI: 77–83), respectively. VE for preventing hospitalization but not medical attention remained stable over 2 years. Vaccinated were less often hospitalized (23%) than unvaccinated RV-infected children (61%; P < 0.001). Breastfeeding (odds ratio, 3.99; 95% CI: 1.92–8.27) and attending daycare (odds ratio, 3.42; 95% CI: 1.64–7.12) were independently associated with breakthrough infections. Genotype G1P[8] was detected more frequently in RotaTeq-vaccinated (44% versus 11%; P < 0.03) and G2P[4] in Rotarix-vaccinated children (42% versus 6%; P < 0.02). Conclusions: RV vaccination protects young children effectively from RV disease and can reduce disease severity. Breastfeeding might impair VE, but further research is needed to identify the critical time window for this interference and to develop appropriate recommendations.

Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR

BackgroundThe principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses.ResultsStool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis.ConclusionsIn this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.

Use of sequence analysis of the P2 domain for characterization of norovirus strains causing a large multistate outbreak of norovirus gastroenteritis in Germany 2012.

Human norovirus is the main cause of non-bacterial gastroenteritis worldwide. It is transmitted from person to person, by fecally contaminated food or water or through virus containing aerosols originating during vomiting of infected persons. In September and October 2012, the largest foodborne norovirus outbreak in Germany so far spread over 5 Federal States (Berlin, Brandenburg, Saxony, Saxony-Anhalt, and Thuringia) affecting nearly 11,000 people mainly in schools and child care facilities. Epidemiological and trace-back investigations supported the assumption that a batch of frozen strawberries imported from China was the likely source of the outbreak. Sequence analysis of the capsid region encoding the P2 domain was used successfully for identification of transmission routes and epidemiologic relationship but was hampered by a lack of universal primers for all known genotypes so far. In the present study, a molecular approach was designed to track outbreak-related samples from the affected states of the large foodborne outbreak in Germany. Therefore, sequence analysis within the highly variable P2 domain of the capsid gene using newly developed universal P2 primers for genogroup I and genogroup II strains in combination with sequencing of the polymerase gene (region A) and the orf1/orf2 junction (region c) was used. The sequence analysis of 138 norovirus positive stool samples suspected to be outbreak-related revealed a considerable genomic diversity. At least 3 strains of genogroup I (I.3, I.4, and I.9) and 5 strains of genogroup II (II.6, II.7, II. 8, and recombinants II.P7_II.6, and II.P16_II.13) as well as 19 samples containing mixtures of these strains were detected. Six samples were considered as not linked to the outbreak. The most prevalent genotype was GI.4 (48/132; 36%). Genotype I.9 and the recombinant strain II.P16_II.13 were detected for the first time in Germany. Notably, the genotype II.P16_II.13 could also be determined in one of the samples of the frozen strawberry lot suspected as infection source. Especially, due to the good concordance of the P2 sequences from infected patients of 5 Federal States the outbreak-relation of the strains could be demonstrated. The high diversity of virus strains and the occurrence of sub-clusters within genotypes I.3, II.8, II.P16_II.13, and II.7 revealed the complex mixture of the outbreak source suggesting a possible waterborne fecal contamination of the strawberries. The typing system described here is in general useful for analysis of outbreaks caused by mixed infection sources. Extensive sequence analysis of different gene regions including the highly variable P2 domain in a sufficient number of cases is required to confirm the epidemiological relation of samples from outbreaks with high diversity of strains spreading over several geographic locations.

Apolipoprotein E allele frequencies in chronic and self-limited hepatitis C suggest a protective effect of APOE4 in the course of hepatitis C virus infection.

Infectious hepatitis C virus (HCV) particles bind to host lipoproteins such as low‐density lipoproteins (LDLs). Low‐density lipoprotein receptors (LDLR) have been termed candidate receptors for HCV–LDL complexes. Functional host genetic single nucleotide polymorphisms (SNPs) in the apolipoprotein E (APOE) gene encoding apolipoprotein E (apoE) – a major structural LDL component and natural ligand of LDLR – likely influence the course of HCV infection. We investigated the prevalence of APOE SNPs in two large and independent cohorts of patients with chronic HCV infection compared to respective controls.

Infection with the Persistent Murine Norovirus Strain MNV-S99 Suppresses IFN- Beta Release and Activation of Stat1 In Vitro

Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-β production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-β release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response.

Group A rotaviruses circulating prior to a national immunization programme in Nigeria: Clinical manifestations, high G12P[8] frequency, intra-genotypic divergence of VP4 and VP7.

Nigeria having approximately 50 000 Rotavirus A (RVA) deaths annually is yet to introduce RVA vaccine into routine national immunization; therefore surveillance of RVA strains circulating before vaccine introduction is essential in evaluating impact of the intervention. Stool samples and sociodemographic data of diarrhoeic children, <5 years were collected between August 2012 and December 2013. While a high prevalence of RVA infection (47.6%; 49/103) was observed by quantitative reverse transcription real time PCR, only 25% (26/103) had high RVA genome concentrations and were antigen positive. G and P types were obtained for 31 and 37 samples respectively. G12P[8] strains were predominant (30.6%; 16/31); Other genotypes found included G9, G3, G2 and P[4], P[6], P[8]. A G12 + G2/P[8] + P[6] mixed infection was detected. The P[8] genotype showed divergence with strains distributed in lineage III and IV. Compared to the vaccines, changes in antigenic sites of VP8* and VP7 were found. The finding of the G2P[6] genotype combination and emergence of G12 strains support observations in most of the recent RVA studies from Africa. P[6] is common in many African countries, in contrast to countries in Europe and the Americas. In conclusion, this study shows the circulation of other RVA genotypes compared to the common RVA genotypes in Nigeria. PCR results should be interpreted with caution to avoid significant bias from samples with low RVA genome concentrations. These findings provide important information on the detection and molecular epidemiology of RVA prior to vaccination and contribute as a baseline for future evaluations after possible vaccine introduction.

Co-circulation of classic and novel astrovirus strains in patients with acute gastroenteritis in Germany

OBJECTIVES In order to analyze the molecular epidemiology of human astroviruses (HAstV) in Germany, a retrospective long-term study was performed to characterize circulating human astrovirus in patients with acute gastroenteritis in Germany. METHODS A total of 2877 stool samples, collected between January 2010 and December 2015 from sporadic cases and outbreaks of acute gastroenteritis were retrospectively analyzed for astrovirus. A two-step PCR algorithm was developed and used to identify and characterize human astrovirus infections. RESULTS Overall, 143 samples were astrovirus-positive (5.0%). Astrovirus infection was most frequently detectable in samples from children of 3-4 years (15%) followed by children of 1-2 years (8.6%), detection rates in adults were lower (1%-3.6%). A high number (71.3%) of co-infections, mainly with noro- or rotaviruses, were identified. Genotyping revealed that at least ten genotypes from all four human MAstV species were circulating in the study population. HAstV-1 was predominant in different age groups. Novel HAstV (MLB and VA genotypes) were also circulating in Germany. CONCLUSION Our findings give new insights into the circulation and genetic diversity of human astroviruses in patients with acute gastroenteritis. The novel HAstV-MLB and -VA genotypes could be characterized firstly in Germany while the analysis showed that these viruses have been dispersed in Germany since 2011 as a causative agent of acute gastroenteritis.

Life-threatening systemic rotavirus infection after vaccination in Severe Combined Immunodeficiency (SCID)

Rotavirus (RV) infections are the major cause of severe gastroenteritis in children under five years of age, causing 215,000 deaths per year worldwide mainly due to dehydration in countries with weak economic resources (1). In the immunocompetent host RV infections are self-limiting and mortality is low in high-income countries. Notifications of RV cases have declined from 49,000 in season 2012/2013 to 25,000 in season 2015/2016 (2) since RV vaccination has been recommended by the Standing Committee on Vaccination (STIKO) in Germany in 2013. The recommendation was based on systematic reviews and meta-analyses of studies showing efficacy and safety of RV vaccination (3). This article is protected by copyright. All rights reserved.